Download Regulation of transcript encoding the 43K

557
Development 104, 00-00 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Regulation of transcript encoding the 43K subsynaptic protein during
development and after denervation
TIMOTHY J. BALDWIN, JULIE A. THERIOT, CORINNE M. YOSHIHARA
and STEVEN J. BURDEN
Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Summary
The postsynaptic membrane of vertebrate neuromuscular synapses is enriched in the four subunits of
the acetylcholine receptor (AChR) and in a peripheral
membrane protein of Mr = 4 3 x l 0 3 (43K). Although
AChRs are virtually restricted to the postsynaptic
membrane of innervated adult muscle, developing and
denervated adult muscle contain AChRs at nonsynaptic regions. These nonsynaptic AChRs accumulate
because the level of mRNA encoding AChR subunits
increases in response to a loss of muscle cell electrical
activity. We have determined the level of mRNA
encoding the 43K subsynaptic protein in developing
muscle and in innervated and denervated adult
muscle. We isolated a cDNA that encodes the entire
protein-coding region of the 43K subsynaptic protein
from Torpedo electric organ and used this cDNA to
isolate a cDNA that encodes the 43K subsynaptic
protein from Xenopus laevis. We used the Xenopus
cDNA to measure the level of transcript encoding the
43K protein in embryonic muscle and in innervated
and denervated adult muscle by RNase protection.
The level of transcript encoding the 43K protein is low
in innervated adult muscle and increases 25- to 30-fold
after denervation. The level of transcript encoding the
alpha subunit of the AChR increases to a similar
extent after denervation. Moreover, during development, transcripts encoding the 43K protein and the
alpha subunit are expressed initially at late gastrula
and are present in similar quantities in embryonic
muscle. These results demonstrate that transcripts
encoding the 43K protein and AChR subunits appear
coordinately during embryonic development and that
the level of mRNA encoding the 43K protein is
regulated by denervation.
Introduction
43K protein is not required for ligand-gated AChR
channel function, since AChR channels retain their
ligand-gated channel characteristics in the absence of
the 43K protein (Neubig et al. 1979; Mishina et al.
1984). The correspondence between the location of
AChR and 43K protein at synaptic sites and the 1:1
stoichiometry of AChR and 43K protein at these sites
has led to the suggestion that the 43K protein is
involved in the formation and/or maintenance of
high-density packing of AChRs at synaptic sites.
Although AChRs are virtually restricted to the
postsynaptic membrane in innervated adult muscle,
developing muscle and denervated adult muscle contain AChRs at their nonsynaptic surface (for reviews,
see Fambrough, 1979; Salpeter, 1987). These AChRs
The 43X103 (43K) protein is a peripheral membrane
protein that is highly concentrated at vertebrate
neuromuscular synapses (for reviews, see Froehner,
1986; Burden, 1987). The 43K protein is a major
protein of the postsynaptic apparatus, since it is
present at 1:1 stoichiometry with the acetylcholine
receptor (AChR) (Burden et al. 1983; LaRochelle &
Froehner, 1986). Moreover, chemical cross-linking
experiments demonstrate that the 43K protein is in
close physical proximity to the cytoplasmic domain(s)
of the beta subunit of the AChR and raise the
possibility that the 43K protein directly interacts with
the AChR (Burden et al. 1983). Nevertheless, the
Key words: neuromuscular synapse, denervation,
peripheral membrane protein, postsynaptic membrane,
Xenopus laevis, Torpedo.
558
T. J. Baldwin
are synthesized and accumulate at nonsynaptic regions in response to a lack of muscle cell electrical
activity. Denervation causes a 10- to 100-fold increase
in AChR mRNA and AChR protein (for review, see
Anderson, 1987). These nonsynaptic AChRs are not
clustered at high density, but rather are present at 20to 100-fold lower concentration than at the synapse.
Although fluorescence and histochemical methods
have been used to detect AChRs that are clustered at
synapses, these methods are not sufficiently sensitive
to detect readily the lower density of nonsynaptic
AChRs in developing and denervated muscle. The
lower density of nonsynaptic AChRs can be detected,
however, by electrophysiological methods and by
autoradiography. Moreover, the high affinity and
specificity of alpha-bungarotoxin binding allows
measurement of AChR protein in detergent extracts
of denervated muscle (for review, see Fambrough,
1979).
Similarly, antibodies against the 43K protein have
been used to detect the 43K protein at synapses, but
these immunochemical methods are not sufficiently
sensitive to determine whether the 43K protein is
present at nonsynaptic regions of denervated and
developing muscle (Froehner et al. 1981; Burden,
1985; Peng & Froehner, 1985). Thus, it is not clear
whether the 43K protein is associated with both
synaptic and nonsynaptic AChRs or whether the 43K
protein is associated with only clustered AChRs.
Moreover, it is not clear whether muscle cell electrical activity regulates the level of 43K protein as
electrical activity regulates the level of AChR.
Since methods for measuring low levels of 43K
protein are not presently available, we have used a
nucleic acid probe to measure the level of transcript
encoding the 43K protein in developing muscle, and
in innervated and denervated adult muscle. We
demonstrate that the level of transcript encoding the
43K protein corresponds to the level of transcript
encoding the AChR in developing muscle, and in
innervated and denervated adult muscle: denervation
results in a 25- to 30-fold increase in the level of
transcript encoding the 43K protein and a 30- to 35fold increase in the level of transcript encoding the
alpha subunit of the AChR. Moreover, during development, AChR mRNA and 43K protein mRNA are
expressed initially at late gastrula and are present at
similar amounts in developing muscle. These results
demonstrate that transcripts encoding the major proteins of the postsynaptic membrane appear coordinately during embryonic development. Moreover, the
level of mRNA encoding the 43K protein is regulated
by denervation and suggests that the level of mRNA
encoding the 43K protein is regulated by myofibre
electrical activity.
Materials and methods
Isolation of cDNA encoding the 43K subsynaptic
protein from Torpedo electric organ
A lambda gtll cDNA library was prepared from Torpedo
electric organ poly(A)+ RNA. The RNA was copied into
cDNA as described (Gubler & Hoffman, 1983; Baldwin et
al. 1988), except that first-strand synthesis was primed with
random primers.
200000 recombinant phage were screened with a nicktranslated (Rigby et al. 1977) cDNA 32P-probe encoding a
truncated form of the Torpedo 43K subsynaptic protein.
This cDNA was generously provided by Drs Frail and
Merlie (Washington University School of Medicine, St
Louis, MO) and has been designated T43k.l (Frail et al.
1987). Filters were hybridized with 32P-probe in 5xSSPE,
5xDenhardt's, 100^gmP 1 calf thymus DNA, and 0-1%
SDS at 68°C and washed in 0-lxSSC, 0 4 % SDS at 68°C
(Benton & Davis, 1977; Maniatis et al. 1982).
Using these procedures, we detected 90 positive plaques,
purified phage from 24 plaques and analysed DNA from
these phage by Southern blots. Phage DNA was digested
separately with EcoRl and Stul and Southern blots were
probed with 32P-T43k.l cDNA. Based upon the restriction
maps of cDNA clones that encode an incorrect C-terminal
region (T43k.l) and a second cDNA clone, which is
incomplete (T43k.7), but encodes the correct C-terminal
region of the 43K subsynaptic protein (Frail et al. 1987), a
1051 bp fragment was predicted to span from a Stul site at
amino acid 80 to a Stul site 68bp 3' to the C-terminus. We
detected two phage that harboured cDNA inserts that
contained a 1051 bp Stul fragment; one cDNA insert is
2-5 kb in length and the other is 1-8 kb in length. The 1-8 kb
cDNA was mapped with restriction endonucleases and
approximately 300 bp from the 5' and 3' ends were
sequenced. The restriction map of the cDNA (Tor 43K)
was equivalent to a spliced product of T43k.l and T43k.7.
Isolation of cDNA encoding the 43K subsynaptic
protein from Xenopus laevis
200000 phage from a lambda gtlO cDNA library prepared
from Xenopus embryo poly(A)+ RNA isolated from stage22 to -24 embryos (Kintner & Melton, 1987; Baldwin et al.
1988) were screened with a nick-translated cDNA 32Plabelled probe encoding the Torpedo 43K protein (see
above). Filters were hybridized with 32P-probe in 5xSSPE,
5xDenhardt's, 100^gml"1 calf thymus DNA, and 0-1%
SDS at 52°C and washed in 0-5xSSC, 0-1 % SDS at 52°C
(Maniatis et al. 1982). These hybridization and wash conditions were established with Northern blots of Xenopus
embryo RNA. Using these procedures, we detected one
positive plaque and this phage was purified (Xen 43.1). The
cDNA insert was subcloned into plasmid vectors, mapped
with restriction endonucleases and sequenced (Baldwin et
al. 1988).
Northern blots and RNase protection
Total RNA was isolated from Xenopus embryos using
proteinase K/SDS as previously described (Baldwin et al.
1988). Total RNA was isolated from adult muscle using
Regulation of 43K subsynaptic protein
guanidinium isothiocyanate as described (Chirgwin et al.
1979). The triceps femoris muscle was denervated as
described previously (Baldwin et al. 1988). Poly(A)+ RNA
was isolated as described and was used for Northern blots
(Baldwin et al. 1988). Hybridization with nick-translated
full-length cDNA ^P-labelled probes was in 5xSSPE,
5xDenhardt's, 100/igmr 1 calf thymus DNA, 0-1% SDS
at 65 °C. Hybridization with 32P-labelled RNA probe was in
50% formamide, 5xSSPE, 5% SDS at 56°C. Filters were
washed in 0-lxSSC, 0-1% SDS at 68°C (Maniatis et al.
1982).
Total cellular RNA was used in RNase protection assays
(Baldwin et al. 1988). The 32P-43K RNA probe was
synthesized with SP6 polymerase from SP65-Xen 43.1
cDNA (3' EcoBJ to 5' EcoRl) linearized with HindlU
(nucleotide 791; Fig. 1). The probe was 448 nucleotides
559
long; 403 nucleotides were protected by cellular and synthetic sense RNA. The 32P-alpha subunit probe was 449
nucleotides long; 429 nucleotides were protected by cellular
and synthetic sense RNA (Baldwin et al. 1988). Purification
of probes, hybridization, digestion and analysis of protected labelled products was as described (Baldwin et al.
1988).
Results
Isolation and characterization of cDNA encoding the
Torpedo 43K subsynaptic protein
Two cDNA clones that encode a portion of the 43K
subsynaptic protein were isolated from a Torpedo
Xenopus laevis 43K subsynaptic protein
5'
CCCTTCCCTGTTCAATTCAATTACTGCCCTAACTCCTGAGCTGCCCA -159
TTTCCCAAACAGGACCACCAGTCATCCTGTCTCTCATGCCAAGCCTTGAGTGAAACCCTCCATTCTTTGGACGCTGAGC
"80
TTCCTAATGTTTTGTAAGCCAGACGGGCTCTGCAGTGGCTCTGCATACCCAGGATTATATGAGACTTTCTGGTGCCGCG
"'
1
20
HET GLY GLN ASP GLN THR LYS GLN GLN ILE GLN LYS GLY LEU GLN MET TYR GLN SER ASN
ATG GGT CAG GAC CAA ACC AAA CAG CAG ATC CAA AAG GGC CTT CAG ATG TAT CAG TCC AAC
60
40
GLN THR GLU LYS ALA LEU GLN ILE TRP THR LYS VAL LEU GLU LYS THR THR ASP ALA ALA
CAG ACA GAG AAG GCT TTG CAG ATC TGG ACT AAA GTC TTG GAG AAG ACC ACT GAT GCG GCC
'20
60
GLY ARG PHE ARG VAL LEU GLY CYS LEU ILE THR ALA HIS SER GLU HET GLY ARG TYR LYS
GGG AGG TTC CGG GTT CTT GGC TGC CTG ATC ACG GCC CAC TCG GAG ATG GGA AGA TAC AAG
'80
80
ASP MET LEU LYS PHE ALA VAL ILE GLN ILE ASP THR ALA ARG GLU LEU GLU GLU PRO ASP
GAT ATG TTA AAG TTT GCA GTG ATC CAA ATC GAC ACG GCT CGG GAG CTG GAG GAG CCA GAC
240
100
PHE LEU THR GLU SER TYR LEU ASN LEU ALA ARG SER ASN GLU LYS LEU CYS GLU PHE GLN
TTT TTG ACC GAG AGT TAC CTC AAC CTG GCC CGT AGC AAC GAG AAG CTC TGC GAG TTC CAG
300
120
LYS THR ILE SER TYR CYS LYS THR CYS LEU ASN MET GLN GLY THR SER VAL SER LEU GLN
AAA ACC ATT TCC TAC TGC AAG ACC TGC CTC AAT ATG CAG GGA ACC TCG GTC AGC CTC CAG
360
HO
LEU ASN GLY GLN VAL CYS LEU SER LEU GLY ASN ALA TYR LEU GLY LEU SER VAL PHE GLN
CTA AAC GGA CAG GTG TGC CTG AGT CTG GGC AAT GCC TAC CTG GGC CTT AGC GTC TTC CAG
420
160
LYS ALA LEU GLU CYS PHE GLU LYS ALA LEU ARG TYR ALA HIS ASN ASN ASP ASP LYS HET
AAA GCC CTG GAA TGC TTC GAG AAG GCC CTG CGC TAC GCC CAC AAC AAC GAT GAC AAG ATG
480
180
LEU GLU CYS ARG VAL CYS CYS SER LEU GLY GLY LEU TYR THR GLN LEU LYS ASP LEU GLU
CTG GAG TGC AGG GTC TGC TGC AGC CTG GGA GGT CTC TAC ACT CAA CTT AAG GAT CTG GAG
540
200
LYS ALA LEU PHE PHE PRO CYS LYS ALA ALA GLU LEU VAL ASN ASP TYR GLY LYS GLY TRP
AAA GCG CTC TTC TTC CCA TGC AAG GCG GCA GAG CTG GTG AAT GAC TAC GGG AAA GGC TGG
600
220
SER LEU LYS TYR ARG ALA MET SER GLN TYR HIS MET ALA VAL ALA TYR ARG LYS LEU GLY
AGC CTC AAA TAC AGA GCG ATG AGT CAG TAC CAC ATG GCG GTC GCT TAC CGC AAG TTG GGC
660
240
ARG LEU ALA ASP ALA MET GLU CYS CYS GLU GLU SER HET LYS ILE ALA LEU GLN HIS GLY
CGT TTA GCC GAC GCA ATG GAG TGT TGT GAG GAG TCA ATG AAG ATC GCC CTT CAG CAT GGA
720
260
ASP ARG PRO LEU GLN ALA LEU CYS LEU LEU ASN PHE ALA ASP ILE HIS ARG SER HIS GLY
GAC CGA CCG CTT CAA GCC CTT TGT CTG CTC AAC TTT GCC GAT ATC CAC AGA AGT CAC GGT
780
280
ASP ILE GLU LYS ALA PHE PRO ARG TYR ASP SER SER HET SER ILE MET THR ASP ILE GLY
GAC ATT GAG AAA GCT TTT CCC CGC TAC GAC TCC TCC ATG AGT ATC ATG ACT GAC ATC GGT
840
300
ASN ARG LEU GLY GLN THR HIS VAL MET ILE GLY VAL ALA LYS CYS TRP LEU HIS GLN LYS
AAC CGC CTG GGT CAG ACC CAT GTA ATG ATA GGA GTG GCG AAA TGT TGG CTC CAT CAG AAG
900
320
GLU MET ASP LYS ALA LEU ASP CYS LEU GLN LYS THR GLN GLU LEU ALA GLU ASP ILE GLY
GAG ATG GAC AAG GCT CTG GAT TGT CTC CAA AAG ACC CAA GAG CTG GCG GAA GAT ATT GGA
960
340
TYR LYS HIS CYS LEU LEU LYS VAL HIS CYS LEU SER GLU ILE ILE PHE ARG THR LYS GLN
TAT AAG CAC TGC CTG CTG AAA GTT CAC TGC CTG AGT GAG ATT ATA TTC CGG ACA AAG CAG
1020
360
GLN GLN ARG GLU LEU ARG ALA HIS VAL VAL ARG PHE HIS GLU CYS VAL GLU GLU MET GLU
CAG CAA CGC GAG CTC CGC GCC CAT GTG GTG CGA TTT CAT GAA TGT GTG GAG GAG ATG GAG
1080
380
LEU TYR CYS GLY HET CYS GLY GLU SER ILE GLY GLU LYS ASN CYS GLN LEU GLN ALA LEU
TTA TAC TGT GGA ATG TGT GGG GAG TCC ATT GGG GAG AAG AAC TGC CAA CTT CAG GCA CTT
1 140
399
PRO CYS SER HIS VAL PHE HIS LEU ARG CYS LEU GLN THR ASN GLY THR ARG GLY CYS
CCG TGC TCC CAT GTC TTT CAT CTG CGG TGT CTT CAG ACC AAT GGA ACC CGA GGC TGC G
1198
Fig. 1. Nucleotide sequence and
deduced amino acid sequence of the
43K subsynaptic protein of Xenopus
laevis. Nucleotide 1 indicates the first
nucleotide of the codon encoding the
amino terminal residue and
nucleotides to the 5' side of this
amino terminal residue are indicated
with negative numbers. The number
of the nucleotide residue at the end
of each line is provided. The
predicted amino acid sequence is
shown above the nucleotide
sequence. Amino acid residues are
numbered beginning with the amino
terminal residue. The 5' and 3' ends
of the Xen 43.1 cDNA is bordered
by EcoRI linkers which were
introduced during cloning.
560
T. J. Baldwin
electric organ cDNA library (Frail et al. 1987). One
cDNA clone encodes a protein whose amino acid
sequence corresponds to the sequence of the 43K
subsynaptic protein from Torpedo electric organ,
except that the cDNA encodes a different C-terminus
and does not encode the last 23 amino acids of the
43K subsynaptic protein (Carr et al. 1987). A second
cDNA clone encodes 42 amino acid residues which
correspond to the C-terminal region and C-terminal
residue of the 43K protein; this cDNA clone, however, is incomplete and 370 of the 412 amino acid
residues are not encoded by the cDNA (Frail et al.
1987). We sought a cDNA that encodes the entire
43K subsynaptic protein from Torpedo electric organ.
Our strategy for isolating this cDNA is described in
Materials and methods.
We isolated a cDNA (Tor 43K) that encodes the
entire protein-coding region of the 43K subsynaptic
protein from Torpedo electric organ. The cDNA is
1-8 kb in length, contains 38 bp of the 5' untranslated
region, the entire protein-coding region that corresponds to the protein sequence of the Torpedo 43K
subsynaptic protein (Carr et al. 1987) and approximately 520 bp of the 3' untranslated region (see
Materials and methods; Fig. 2). The Tor 43K cDNA
was used as a probe to screen a Xenopus embryo
cDNA library.
Isolation and characterization of cDNA encoding the
Xenopus 43K subsynaptic protein
We isolated a cDNA clone encoding the Xenopus
43K protein by screening a Xenopus laevis embryo
cDNA library with a cDNA clone encoding the
Torpedo electric organ 43K protein (see above and
Materials and methods). The sequence of the cDNA
was determined and both the nucleic acid and deduced amino acid sequences were compared to that
for the Torpedo 43K protein (Figs 1 and 2). Xen 43.1
cDNA is 1403 bp long, contains 205 bp of the 5'
untranslated region and 1198 bp of protein-coding
region. Comparison of the deduced amino acid sequence with the sequence of the Torpedo 43K protein
indicates that Xen 43.1 cDNA ends prior to a
termination codon and that the last 13 amino acids of
the Xenopus 43K protein are not encoded by Xen
43.1 cDNA (Figs 1 and 2). Xen 43.1 cDNA does,
however, encode 10 amino acids beyond the Cterminus encoded in a truncated Torpedo 43K clone
(Frail et al. 1987). Since Northern blots of Xenopus
embryo RNA demonstrate that the cDNA clone
hybridizes to transcripts of 4-0kb and 2-0 kb (Fig. 3;
see below), the Xenopus cDNA clone is not fulllength.
The Xenopus 43K protein is 71 % homologous to
the Torpedo 43K protein (Fig. 2). The DNA sequences within the protein-coding region are 72%
homologous. Moreover, the similarity in amino acid
sequence is rather evenly distributed over the length
of the protein (Fig. 2). It is noteworthy that the
homology between the Xenopus and Torpedo sequences does not extend 5' to the codon encoding the
methionine that has been designated as the /V-terminus; since the TV-terminal sequence of the Torpedo
43K protein has not been established (Carr et al.
1987), the initiator methionine was assigned on the
basis of other criteria (Frail et al. 1987). The dissimi-
Alignment of the amino acid sequences of the 43K protein from Xenopus and Torpedo
XN43
TOR43
MGQDQTKQQIQKGLQMYQSNQTEKALQIWTKVLEKTTDAAGRFRVLGCLITAHSEMGRYKDMLKFAVIQIDTARELEEPD
E
L—A-E-G
E—QQ-V-RS-ELP
A
K-E
R
A-SEA—QMGD-E
10'
20'
30*
40*
50"
607080-
XN43
TOR4 3
FLTESYLNLARSNEKLCEFQKTISYCKTCIJmQGTSVSLQLNGQVCLSLGNAYLGLSVFQKALECFEKALRYAHNNDDKM
RV—A
GH
SEAVA—R
GAE-GPLR—F
M
F
A
G
90100110120130140150160-
XN43
TOR43
LECRVCCSLGGLYTQLKDLEKALFFPCKAAELVNDYGKGWSLKYRAMSQYHMAVAYRKLGRLADAMECCEESMKIAIiQHG
AF-V
Y
S
A—R
K—R
A
MD
Q
170180'
190200210220230'
240 A
XN43
TOR43
DRPLQALCLLNFADIHRSHGDIEKAFPRYDSSHSIHTDIGNRLGQTHVMIGVAKCWLHQKEMDKALDCLQKTQELAEDIG
C
HRS—G--L
E--LN
E
A--LLNI
MTE-KL--T-GW--AE
DAV250"
260270280290"
300310320"
XN43
TOR43
YKHCLLKVHCLSEI1FRTKQQQRELRAHWRFHECVEEMELYCGMCGESIGEKNCQLQALPCSHVFHLRCLQTNGTRGC
N-LV
A
Y-T-Y-EMGSDQL—D
K
M-D
L
DQ-S
L
K
N
P
330340350360370380390400-
TOR43
NCKRSSVKPGYV
410-
Fig. 2. Alignment of the amino acid sequences of the Xenopus laevis and Torpedo californica 43K proteins. The
Xenopus (XN 43) sequence is shown by the one-letter amino acid notation. Identical residues in the Torpedo (TOR 43)
sequence are indicated with a dash (—) and amino acid substitutions are shown by the one-letter amino acid notation.
71 % (282/399) of the amino acid residues in the two sequences are identical.
Regulation of 43K subsynaptic protein
larity in sequence between the Xenopus and Torpedo
clones in the region 5' to this methionine codon and
the striking similarity in sequence thereafter provides
support for the correct identification of the initiator
methionine. In addition, a glycine residue that is a
putative yV-terminal myristylation addition site
(amino acid residue number two) is conserved in
Xenopus.
cDNA encoding the 43K protein hybridizes to 4-0 kb
and 2-0 kb transcripts in Northern blots of Xenopus
embryo RNA
Northern blots of poly(A)+ RNA from Xenopus
embryos and denervated adult muscle were probed
with either nick-translated Xen 43.1 cDNA 32Plabelled probe or a 32P-labelled RNA probe encoding
the C-terminal region of the 43K protein (Materials
and methods). Northern blots of embryo RNA and
denervated adult muscle RNA are identical: strong
hybridization is detected to a 4-0 kb transcript and
less intense hybridization to a 2'0kb transcript
(Fig. 3). We do not know whether the 2-0kb transcript is less abundant than the 4-0 kb transcript
and/or whether the 2-0 kb transcript is less homologous to the Xen 43.1 cDNA. Moreover, we cannot
exclude the possibility that the 2-0 kb transcript is a
degradation product of the 4-0 kb transcript.
Fig. 3. cDNA encoding the 43K
protein hybridizes to transcripts of
4-0 kb and 2-0 kb in Northern blots
of Xenopus embryo RNA. 1 n% of
poly(A)+ RNA from stage-41
Xenopus embryos was fractionated
by electrophoresis in a formaldehyde
agarose (1%) gel, transferred to
Zetabind, and hybridized to 32Plabelled Xen 43.1 RNA probe
(Materials and methods). The RNAs
that hybridize migrate at 40 and
2-0kb (arrowheads). The filter was
exposed to X-ray film with an
intensifying screen at —70°C for
lday.
43K
INN
DEN
561
AChR
INN DEN
Fig. 4. Transcript encoding the 43K protein and
transcript encoding the AChR alpha subunit are 25- to
35-fold more abundant in denervated than in innervated
adult muscle. The triceps femoris muscle of adult
Xenopus was denervated for 10 days and AChR alpha
subunit and 43K protein transcript levels were measured
by RNase protection. 5 fig of RNA from innervated
(INN) and denervated (DEN) muscle were included in
hybridizations with 32P-labelled probes. The amount of
alpha subunit and 43K protein transcript is low in
innervated muscle and increases 25- to 35-fold in
denervated muscle. Denervation results in no change in
either the amount of total RNA or the level of transcript
encoding the translation elongation factor Ef-1-alpha
(Baldwin et al. 1988). The arrowheads mark the positions
of the protected fragments at 403 nucleotides (43K
protein) and 429 nucleotides (AChR alpha subunit).
Transcript encoding 43K protein is 25- to 30-fold
more abundant in denervated than in innervated
adult muscle
We isolated total cellular RNA from innervated adult
Xenopus muscle and denervated adult Xenopus
muscle and measured the level of transcripts encoding
the 43K protein and the alpha subunit of the AChR.
We had demonstrated previously that transcript encoding the alpha subunit increases 50- to 100-fold
after denervation of Xenopus muscle, whereas denervation results in no change in either the amount of
total RNA or transcript encoding the translation
elongation factor Ef-1-alpha (Baldwin et al. 1988).
Fig. 4 demonstrates that alpha subunit transcript
level increases 30- to 35-fold and 43K protein transcript level increases 25- to 30-fold after denervation.
Since both the 2-0 and 4-0 kb RNAs hybridize to the
562
T. J. Baldwin
12
14
20
41
Fig. 5. Transcript encoding the 43K protein is first expressed at late gastrula. 43K protein transcript levels were
measured in Xenopus embryos by RNase protection. RNA from 30 eggs (E) and 30 embryos at stages 10, 12 and 14
were included in hybridizations with 32P-labelled Xen 43 probe. Hybridization reactions with RNA from embryos at
later stages (20 and 41) included RNA from 10 embryos. The gel was exposed to X-ray film with an intensifying screen
at —70°C for 4 days to analyse expression at early stages (egg, 10, 12 and 14) and for 3 days to analyse later stages (20
and 41). The stages are indicated at the top of each lane; the arrowhead marks the position of the protected fragment at
403 nucleotides.
encoding the subsynaptic 43K protein increases 25- to
30-fold after denervation of adult skeletal muscle.
Denervation of adult muscle produces a similar
increase in mRNA encoding AChR subunits (Merlie
et al. 1984; Evans et al. 1987; Moss et al. 1987;
Baldwin et al. 1988). Thus, loss of myofibre electrical
activity results in a 25- to 100-fold increase in the level
Transcript encoding 43K protein is expressed initially of transcripts encoding the major proteins of the
at late gastrula during embryonic development
postsynaptic membrane.
To determine when transcript encoding the 43K
Although denervation results in a similar increase
protein is first expressed during embryonic developin AChR mRNA and AChR protein (Merlie et al.
ment, we isolated total cellular RNA from Xenopus
1984), we have not measured the level of 43K protein
embryos and measured the level of transcript enin innervated or denervated muscle. Nevertheless,
coding the 43K protein. Fig. 5 demonstrates that
the results presented here suggest that the level of
transcript encoding the 43K protein isfirstdetected in
43K protein increases following denervation and raise
Xenopus embryos at late gastrula (stage 12); tranthe possibility that the 43K protein is associated with
script is not detected in eggs or in stage-10 embryos.
AChRs that are neither clustered nor present at
At later stages of development transcript encoding
synaptic sites.
the 43K subsynaptic protein is more abundant
Transcript encoding the 43K protein is readily
(Fig. 5). Initial expression of AChR subunit trandetected in Xenopus embryos at stage 14 (16-25 h of
scripts also occurs at stage 12 and similar increases in
development) and is first detected at stage 12
AChR subunit transcript levels occur during later
(13-75 h). Transcripts encoding AChR subunits are
stages of development (Baldwin et al. 1988). Morealso initially expressed at stage 12 of development
over, the absolute quantity of transcripts encoding
(Baldwin et al. 1988). Moreover, the amount of
AChR subunits and transcript encoding 43K protein
transcript encoding the 43K protein is similar to that
is similar at all stages. Thus, expression of transcripts
encoding AChR subunits throughout development
encoding AChR subunits and the 43K protein are
(Baldwin et al. 1988). Thus, transcript encoding the
regulated in a similar manner during development as
43K protein is present before synapses form (stage 21,
well as in innervated and denervated adult muscle.
22-5 h) (Kullberg et al. 1977) and before AChRs
cluster at synapses (stage 22, 24 h) (Chow & Cohen,
1983). Thus, synapse formation and clustering of
Discussion
AChRs are not required to initiate transcription of
the gene encoding the 43K protein.
This study demonstrates that the level of transcript
43K RNA probe at high stringency (Fig. 3), the
RNase protection analysis measures the sum total of
each transcript. Thus, the level of transcripts encoding both the alpha subunit of the AChR and the
43K protein are low in innervated adult muscle and
increase to a similar extent following denervation.
Regulation of 43K subsynaptic protein
Previous immunocytochemical studies have
demonstrated that the 43K protein is restricted to the
postsynaptic membrane of vertebrate skeletal muscle
cells. The 43K protein has not been detected by
indirect immunofluorescence at synaptic sites on
parasympathetic neurones in the frog cardiac
ganglion or in either plexiform layer of the frog retina
(unpublished data). The availability of a probe that
provides a sensitive assay for the transcript encoding
the 43K protein should provide a different and more
sensitive assay for the expression of the 43K protein
in the nervous system.
We would like to thank Drs Frail and Merlie for
providing us with Tor43k.l cDNA. This work was supported by a grant from the National Institutes of Health
(NS 21579).
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{Accepted 30 July 1988)